This invention relates to novel plate devices for use in high-throughput screening assays.
The binding of small molecules and peptides to protein is a very important parameter to measure in the pharmaceutical industry. As pharmaceutical companies strive to design novel small molecules and peptides to treat various ailments ranging from life threatening diseases including cancer, AIDS, and heart disease to cosmetic complaints such as ACNE, age spots and wrinkles, successful administration of these drugs through the circulatory system is critical. Many drugs that have been shown to be very active in vitro assays have failed to show efficacy in animal models and in people because of the high plasma protein binding exhibited by these compounds. When a molecule is highly bound to proteins in the blood the amount of drug available to diffuse into the target tissue is significantly reduced and the efficacy of the drug will inevitably be poor.
Whether a small molecule binds to plasma proteins or not usually depends on the size of the molecule, the amino acid composition and the tertiary structure of the molecule. When a small molecule binds to plasma proteins the interaction usually is a result of strong ionic and hydrophobic interactions. Because blood contains several hundred proteins there is a high probability that any small molecule will exhibit some level of binding. Determining the level of binding therefore is critical and will directly correlate with efficacy of the molecule. Predicting whether a molecule is going to show high or low protein binding based on molecular structure has proven to be very difficult. The only sure way to determine whether or not a molecule will exhibit high or low protein binding is to test the molecule directly in a protein-binding assay.
The most common method used to measure the level of protein binding exhibited by compounds is equilibrium dialysis assays. In such assays, a set concentration of drug (usually 1xc2x7M) is added to a set volume of human plasma (usually 3 ml). The mixture is added to dialysis tubing with a molecular weight cut-off of 30 kDa. The mixture is allowed to incubate in a large volume of water (usually 4 liter) for 24 hours at 37xc2x0 C. Following the incubation the sample is collected and the concentration of drug is calculated. If the compound is completely unbound to protein, the concentration following dialysis would be 0, if 50% bound the concentration would be 0.5 uM, etc. Although equilibrium dialysis has been shown to be accurate and consistent, it is very time consuming and the number of drugs a researcher can test in one assay is dependent on how many 4-liter beakers he/she can set up. Thus, the need for a fast, high-throughput assay is needed in the pharmaceutical industry.
This invention is directed to a filtrate-holder plate comprising polypropylene and Teflon(copyright), most preferably about 2.5% Teflon(copyright) and about 97.5% polypropylene. Said plate contains 96 wells each of which is capable of securely holding a Microcon-3, 10, 30 or 100(copyright) filter unit (see FIGS. 4, 5, 7 and 8). Said filter unit (see FIG. 1) contains a reservoir and a filter membrane, contained within the filtrate plate. The filter unit also contains a base, which passes through an opening in each of the filtrate plates 96 wells and into a reversible-well collection plate (see FIGS. 9, 10, 11 and 12). Said xe2x80x9creversible-well collection platexe2x80x9d, also comprises polypropylene and Teflon(copyright) (most preferably, again, at about 2.5% Teflon(copyright) and about 97.5% polypropylene) and also contains 96 wells. The reversible-well plate is designed such that its wells can accommodate either end of the Micron-3, 10, 30 or 100(copyright) filter unit.
The filtrate-holder and reversible-well plates, along with the filter units contained therein, are securely fastened together and used in high-throughout screening assays for the binding of small molecules and peptides to proteins. The design of the plates, and their composition, allows for several advantages in the conducting of such screens, as is described herein.